Abstract 13203: Novel Photoreleasable Caged Angiotensin-II Analogs to Study Intracrine Pharmacology in Intact Cells
Background: Angiotensin type 1 (AT1R) and type 2 (AT2R) receptors on intracellular membranes might play an important role in cardiac remodeling, but the importance of intracellular “intracrine” signaling is unknown, largely because of a lack of adequate tools to allow selective activation of intracellular ATRs. Here, we developed and applied photoactivatable caged Ang-II analogs to specifically examine intracrine Ang-II signaling.
Methods and Results: Caged Ang-II analogs were synthesized by incorporating a photo-cleavable 4,5-dimethoxy-2-nitrobenzyl group either on Tyr4 (DMNB), the α-COOH terminal (ODMNB), or both. All three analogs displaced [125I]Ang-II binding and induced contraction upon extracellular administration in aortic ring preparations with 2-3 fold reduced affinity. Mass spectrometry confirmed that UV irradiation of caged Ang-II released native Ang-II (Fig A). Tyr(DMNB)4]Ang-II (cAng-II) showed faster rates of photolysis than ODMNB analogs and was employed for subsequent studies. Whereas native Ang-II (10 nM) and photolysed cAng-II (10 nM) increased ERK-phosphorylation (AT1R) and cGMP production (AT2R), non-irradiated cAng-II (10 nM) did not. We previously showed that Ang-II mobilizes nuclear Ca2+ through IP3-receptor channels and nuclear envelope AT1R in isolated cardiomyocyte nuclei. Hence, we studied the ability of cAng-II to alter nucleoplasmic Ca2+ ([Ca2+]n) in intact cells using a fluorescent Ca2+ dye, Fluo-4. Photolysis of intracellular cAng-II increased [Ca2+]n (Fig B, C) to 171±16 (F/F0) versus 99±6 in control (P<0.01, N=8/group), confirming Ang-II nuclear intracrine signaling for the 1st time in intact cells.
Conclusions: [Tyr(DMNB)4]Ang-II is a novel Ang-II analog that permits the study of intracrine Ang-II signaling in intact cells. The use of caged ligands in vivo with targeted uncaging technologies could provide novel pharmacological approaches to target selectively intracellular Ang-II signaling.
- © 2013 by American Heart Association, Inc.